Composition comprising at least one bifidobacterium strain for the treatment of vitiligo

Abstract

The invention relates to a composition for preventing and / or treating, in a subject, the early stage of vitiligo corresponding to the dysregulation of the mitochondrial function, keratinocytes and melanocytes, associated with an abnormally high level of ROS in these mitochondria, said composition comprising at least one strain of Bifidobacterium longum or Bifidobacterium breve or a lysate thereof.

Description

COMPOSITION COMPRISING AT LEAST ONE STRAIN OF BIFIDOBACTERIUM FOR THE TREATMENT OF VITILIGO

[0001] [This patent application claims priority from French patent application FR 2205567 filed on June 9, 2022 and hereby incorporated by reference. Field of invention

[0002] The present invention relates to compositions and their use in the treatment or prevention of vitiligo. State of the art

[0003] Vitiligo is a skin condition characterized by the appearance of white patches on the hands, feet, face, or any other part of the body. These patches are caused by depigmentation, which is the disappearance of melanocytes. This depigmentation can be more or less severe, and the white patches can vary in size. In some cases, the hair growing within the depigmented areas may also be white.

[0004] Vitiligo is neither contagious nor painful, but it can cause significant psychological distress. It should be noted that people with dark skin suffer from it in particular, as the condition is even more visible in them. However, the impact on quality of life is significant for all those affected. Vitiligo is still insufficiently treated by doctors.

[0005] Vitiligo affects approximately 0.5 to 2% of the world's population, regardless of ethnicity or gender. It typically appears between the ages of 10 and 30, with half of those affected developing it before the age of 20. Vitiligo is therefore quite rare in young children. It affects both men and women.

[0006] There are several types of vitiligo:

[0007] 1) Segmental vitiligo, located on one side of the body, for example on part of the face, upper body, leg or arm. This form of vitiligo appears more often in children or adolescents. This form appears quickly within a few months, then generally stops progressing.

[0008] 2) Non-segmental vitiligo, which appears as patches that are often more or less symmetrical, affecting both sides of the body. The progression is unpredictable, with the patches remaining small and localized or spreading rapidly.

[0009] Most often, the disease progresses at an unpredictable pace and can stop or spread continuously or, more often, in flare-ups. Vitiligo can therefore progress in phases, with worsenings sometimes occurring after a psychological or physical trigger. In very rare cases, the plaques disappear on their own. These people are also more likely to suffer from other autoimmune diseases, including autoimmune thyroid disorders.

[0010] Vitiligo is a multifactorial polygenetic disease resulting in the loss of melanocytes.

[0011] Several hypotheses are currently being put forward to explain the loss of melanocytes. Vitiligo is likely a pathology that has genetic, environmental, and autoimmune origins. Vitiligo is a disease with a strong autoimmune component. The involvement of cellular immunity, particularly the IFN gamma pathway, is now well demonstrated. Innate immunity is also involved and provides the link between external or internal stressors and the activation of adaptive immunity.

[0012] According to several studies, keratinocytes and melanocytes of people with vitiligo initially accumulate numerous free radicals, which then lead to the production of cytokines and chemokines by keratinocytes and melanocytes and the activation of the immune system.

[0013] To date, vitiligo treatments often require many months and are mainly effective for facial lesions. Failures remain numerous, particularly for bony prominences and the hands and feet. The standard treatment combines phototherapy (at best UV-B) with local treatments using dermocorticoids or calcineurin inhibitors. Highly active forms can benefit from mini-pulses of cortisone. Finally, localized and stable forms and segmental vitiligo are good indications for surgical treatment, particularly by epidermal cell suspension grafting.

[0014] There is therefore a strong demand for a more effective, well-tolerated, and more affordable therapy. Topical administration is an alternative, but only small molecules are suitable for penetrating the epidermis and effectively targeting keratinocytes and melanocytes. Detailed description of the invention:

[0015] The inventors have now demonstrated that extracts of bacterial strains belonging to the species Bifidobacterium longum or Bifidobacterium breve exhibited a particularly significant activity in reducing mitochondrial oxidative stress in a cellular model associated with the early stage of vitiligo corresponding to the deregulation of the mitochondrial function of keratinocytes.

[0016] Accordingly, a first aspect of the invention relates to a composition which aims to prevent and / or treat, in a subject, the early stage of vitiligo corresponding to the deregulation of mitochondrial function, keratinocytes and melanocytes, associated with an abnormally high level of ROS within these mitochondria, which composition comprises at least one strain of Bifidobacterium longum or Bifidobacterium breve, or a lysate thereof.

[0017] Patent application FR 2999933 A1 describes the increase in melanin production in primary human melanocytes (NHEM) following the application of an extract of Bifidobacterium longum. Such a property of increasing melanin production in melanocytes effectively makes it possible to consider homogenizing the complexion. However, such a property does not in any way make it possible to consider preventing or treating vitiligo, let alone the early stage of vitiligo. Indeed, this activity has no effect on the disappearance of melanocytes which characterizes this pathology. Conversely, and in light of this teaching, a person skilled in the art would reasonably have avoided using such an extract of Bifidobacterium longum in patients suffering from vitiligo at the risk of further weakening an already weakened cell population (i.e. melanocytes) and accelerating, rather than slowing down, the onset of the pathology.

[0018] Preferably, said composition aims to prevent and / or treat a subject suffering from vitiligo.

[0019] In relation to vitiligo, this can correspond equally well to segmental vitiligo or non-segmental vitiligo.

[0020] As for the subject, it is a mammal and preferably a human.

[0021] Bifidobacterium is a genus of Gram-positive, anaerobic, bifidobacterial actinobacteria. These bacteria consist of irregularly shaped, strictly anaerobic, catalase-negative, and immobile bacilli. Bifidobacteria belong to the lactic acid bacteria family. They participate in the fermentation of milk in the manufacture of cheese and yogurt-like preparations. In addition, they produce large amounts of lactic acid, which lowers the pH, which is favorable to them and is believed to inhibit the growth of other bacteria. The species Bifidobacterium longum or Bifidobacterium breve are present in the human digestive tract.

[0022] Advantageously, the content of the composition according to the invention in at least one strain of Bifidobacterium longum or Bifidobacterium breve is between 10 2 and 10 13 CFU / ml of composition, preferably it is between 105 and 10 11 CFU / ml of composition.

[0023] Now, the said at least one strain of Bifidobacterium longum or Bifidobacterium breve may not be kept viable after its integration into the composition. In this context, the composition may be stored at room temperature.

[0024] Lysate means a culture of such a bacterial strain that has undergone cell lysis.

[0025] Cell lysis can be achieved by multiple technologies such as osmotic shock, thermal shock, ultrasound treatment or mechanical stress (e.g. centrifugation).

[0026] Advantageously, such a lysate is obtained by lysis by thermal shock or by ultrasound.

[0027] Typically, such a lysate can be obtained according to the technology described in US patent No. 4,464,362 and in particular by following the protocol below.

[0028] Microorganisms corresponding to Bifidobacterium species are cultivated under anaerobic conditions in a suitable culture medium (see for example the US Patents No. 4,464,362 and EP 0 043 128 B1). When the stationary growth phase is reached, the cultures are inactivated by pasteurization, for example by a temperature of 60 to 65°C for 30 minutes. The microorganisms are then collected by a conventional separation technique, for example by membrane filtration, centrifugation and resuspension in sterile physiological saline solution. The lysate is then obtained by ultrasonic disintegration of the microorganisms so as to release the cytoplasmic fractions, cell wall fragments and metabolic products. All these components are then stabilized in a weakly acidic solution.

[0029] Preferably, it is a lysate of Bifidobacterium longum (CAS No. 96507-89-0). Examples of such a lysate include the product registered under the name “REPAIR COMPLEX CLR™” by the company K. RICHTER GmbH, the product registered under the name PROMACARE® BFL by the company UNIPROMA or the product registered under the name BIFID AFERM™ by the company THE GARDEN OF NATURAL SOLUTIONS.

[0030] Alternatively, it will be a lysate of Bifidobacterium breve.

[0031] Advantageously, the content of the composition according to the invention in lysate of at least one strain of Bifidobacterium is between 1 and 20% (by weight relative to the total weight of the composition), preferably it is between 5 and 10%.

[0032] Antioxidant means a molecule capable of blocking or slowing down the oxidation of another molecule upon contact.

[0033] This class of molecules includes a large number of components, including vitamins, metallic species, peptides or peptide derivatives, polyphenols, flavonols and organosulfur compounds.

[0034] Vitamins with antioxidant activity include vitamin A, vitamin C, vitamin E, coenzyme Q10 and their derivatives.

[0035] Examples of metal species with antioxidant activity include selenium and zinc.

[0036] Examples of pigments with antioxidant activity include fL carotene, lycopene and curcumin.

[0037] As peptides or peptide derivatives exhibiting antioxidant activity, mention may be made of L-camosine, Glutathione or Superoxide dismutase (SOD). L-camosine is a dipeptide of beta-alanine and histidine available in particular under the name SPECPED®LCS (SPEC-CHEM IND) or DRAGOSINE® (SIMRISE). Glutathione is a pseudo-tripeptide formed by the condensation of glutamic acid, cysteine ​​and glycine which is available in particular under the name SPECKAREE® GSH (SPEC-CHEM IND) or AC-GSH (SOHO ANECO CHEMICALS). Superoxide dismutase (SOD) are metalloproteins catalyzing the dismutation of superoxide anions into oxygen and hydrogen peroxide. SODs are available in particular under the name BIOCELL SOD (LONZA) or DISMUTIN®PF (DSM).

[0038] Polyphenols with antioxidant activity include anthocyanins in red fruits, proanthocyanidins in chocolate, caffeoylquinic and feruloylquinic acids in coffee, flavonoids in citrus fruits, catechins in green tea, and quercetin in apples.

[0039] Examples of flavonols with antioxidant activity include quercetol and PYCNOGENOL®.

[0040] Organosulfur compounds with antioxidant activity include lipoic acid, methionine and cysteine.

[0041] Advantageously, the antioxidant content of the composition according to the invention is between 0.05 and 10% (by weight relative to the total weight of the composition). Indeed, the antioxidant content to be adopted is likely to vary greatly depending on the antioxidant capacity of the chosen molecule.

[0042] Now, the inventors have shown that sinapine and its derivatives also exhibit particularly significant activity in reducing mitochondrial oxidative stress in the same cell model. Therefore, a combination of this antioxidant and the bacterial species (or their lysates) mentioned above is of definite interest for the treatment of this early stage of vitiligo.

[0043] According to a preferred embodiment, the antioxidant is sinapine, a salt thereof, or one of their derivatives.

[0044] Sinapine is an alkaloid with the formula (I)

[0046] By sinapine derivative is meant a compound having the formula (II)

[0048] In which

[0049] - R1 and R3 are independently chosen from a hydrogen atom or a C1 to C8 alkyl group, preferably a C1 to C4 alkyl group and, particularly preferably, a C1 to C2 alkyl group; and

[0050] - R2 is independently chosen from a hydrogen atom or a C1 to C8 alkyl group, preferably a C1 to C4 alkyl group and, particularly preferably, a C1 to C2 alkyl group.

[0051] Sinapine is present in quantity in the seeds or germs of plants of the Brassicaceae family, such as rapeseed (Brassica napus), watercress (Lepidum sativum), mustard (Brassica alba).

[0052] Advantageously, sinapine is in the form of an extract of a seed or germ of a plant belonging to the Brassicaceae family.

[0053] The term "extract" refers to a substance extracted from a natural product, regardless of its extraction method or the composition of the ingredients. For example, this includes those obtained by extracting soluble ingredients from a natural product using water or an organic solvent, or those obtained by extracting only specific ingredients, such as oil, from a natural product. Extraction methods are well known to those skilled in the art and typically consist of macerating the seeds, whether ground or not, in the extraction solvent.

[0054] Preferably, the extract of seeds or germs of a plant belonging to the Brassicaceae family is an aqueous, alcoholic or hydro-alcoholic extract. Advantageously, an extract using glycerin as an extraction solvent will be used, such as glycerin, hydroglycerin or glycerin hydroalcoholic extracts.

[0055] Examples of such extracts include an aqueous extract of mustard sprouts such as that marketed by the company MIBELLE under the name LIPERFECTION or the hydroglycerin extract of watercress sprouts marketed by the company MIBELLE under the name DETOXOPHANE.

[0056] According to a preferred embodiment, the composition according to the invention is intended for topical application to the skin, in particular the skin of the face, hands, neck and body in general. As such, the composition according to the invention takes the form of a cream, a serum, a milk, a lotion or a gel. Preferably, the composition according to the invention will take the form of a cream.

[0057] The composition according to the invention is then applied to the affected skin surface at least once a day, preferably at least twice a day (e.g.: once in the morning and once in the evening).

[0058] In general, the topical composition according to the invention may comprise numerous types of adjuvants or active ingredients used in pharmaceutical or cosmetic formulations, preferably dermatological formulations, whether fatty substances, organic solvents, thickeners, gelling agents, softeners, opacifiers, stabilizers, foaming and / or detergent surfactants, emollients, superfatting agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestering agents, chelators, preservatives, essential oils, coloring materials, pigments, hydrophilic or lipophilic active ingredients, humectants, such as for example glycerin or glycols, preservatives, colorants, cosmetic active ingredients, mineral and / or organic sunscreens, mineral fillers, synthetic fillers, silicone elastomers, or extracts of plants or lipid vesicles, or any other ingredient usually used in cosmetics.

[0059] Now and advantageously, the composition according to the invention will not include paraben.

[0060] Still advantageously, the composition according to the invention will not comprise phenoxyethanol.

[0061] Examples of oils include paraffins, isoparaffins, white mineral oils, vegetable oils (from flowers, fruits, vegetables, trees, cereals, oilseeds, etc.), animal oils, synthetic oils, silicone oils and fluorinated oils;and more particularly: oils of vegetable origin, such as sweet almond oil, coconut oil, castor oil, jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil, wheat germ oil, corn germ oil, soybean oil, cottonseed oil, alfalfa oil, poppy seed oil, pumpkin oil, evening primrose oil, millet oil, barley oil, rye oil, safflower oil, candlenut oil, passionflower oil, hazelnut oil, palm oil, shea butter, apricot kernel oil, calophyllum oil, sisymbra officinalis oil, avocado oil, calendula, oils from flowers or vegetables; ethoxylated vegetable oils; oils of animal origin, such as squalene, squalane; mineral oils, such as paraffin oil, vaseline oil and isoparaffins;synthetic oils, including fatty acid esters such as butyl myristate, propyl myristate, cetyl myristate, isopropyl palmitate, butyl stearate, hexadecyl stearate, isopropyl stearate, octyl stearate, isocetyl stearate, dodecyl oleate, hexyl laurate, propylene glycol dicaprylate;esters derived from lanolic acid, such as isopropyl lanolate, isocetyl lanolate, monoglycerides, diglycerides and triglycerides of fatty acids such as glycerol triheptanoate, alkylbenzoates, polyalphaolefins, polyolefins such as polyisobutene, synthetic isoalkanes such as isohexadecane, isododecane, perfluorinated oils and silicone oils. Among the latter, we can more particularly cite dimethylpolysiloxanes, methylphenylpolysiloxanes, silicones modified by amines, silicones modified by fatty acids, silicones modified by alcohols, silicones modified by alcohols and fatty acids, silicones modified by polyether groups, modified epoxy silicones, silicones modified by fluorinated groups, cyclic silicones and silicones modified by alkyl groups.;

[0062] Other fats include fatty alcohols, linear or branched, saturated or unsaturated, mixtures of fatty alcohols, linear and / or branched, saturated and / or unsaturated, or fatty acids, linear or branched, saturated or unsaturated, mixtures of linear or branched, saturated or unsaturated fatty acids.

[0063] Among the thickening and / or emulsifying polymers that can be used, there are, for example, homopolymers or copolymers of acrylic acid or acrylic acid derivatives, homopolymers or copolymers of methacrylic acid or methacrylic acid derivatives, homopolymers or copolymers of acrylamide, homopolymers or copolymers of acrylamide derivatives, homopolymers or copolymers of acrylamidomethyl propanesulfonic acid, homopolymers or copolymers of vinyl monomers, homopolymers or copolymers of trimethylaminoethylacrylate chloride, hydrocolloids of plant or biosynthetic origin such as, for example, xanthan gum, karaya gum, carrageenans, alginates; silicates; cellulose and its derivatives; starch and its hydrophilic derivatives; polyurethanes.

[0064] Among the polyelectrolyte polymers that may be used in the production of a gelled aqueous phase suitable for use in the preparation of W / O, O / W, W / O / W or O / W / O emulsions, or of an aqueous gel comprising SEPIBIO™ POTENTILLA 217, there are, for example, copolymers of acrylic acid and 2-methyl-[(l-oxo-2-propenyl)amino] 1 -propane sulfonic acid (AMPS), copolymers of acrylamide and 2-methyl-[(l-oxo-2-propenyl)amino] 1 -propane sulfonic acid, copolymers of 2-methyl-[(l-oxo-2-propenyl)amino] 1 -propane sulfonic acid and (2-hydroxyethyl)acrylate, homopolymer of 2-methyl-[(l-oxo-2-propenyl)amino]-1-propanesulfonic acid, acrylic acid homopolymer, copolymers of acryloyl ethyl trimethyl ammonium chloride and acrylamide, copolymers of AMPS and vinylpyrrolidone, copolymers of AMPS and N,N-dimethylacrylamide., terpolymers of AMPS, acrylic acid and N,N-dimethylacrylamide, copolymers of acrylic acid and alkyl acrylates whose carbon chain comprises between ten and thirty carbon atoms, copolymers of AMPS and alkyl acrylates whose carbon chain comprises between ten and thirty carbon atoms.

[0065] Among the waxes that can be used in the compositions according to the invention, mention may be made, for example, of beeswax, camauba wax, Candelilla wax, Ouricoury wax, Japan wax, China wax, rice bran wax, Montan wax, cork or sugarcane fiber wax, paraffin waxes, lignite waxes, microcrystalline waxes, lanolin wax, ozokerite, polyethylene wax, hydrogenated oils, silicone waxes, alkenone waxes, vegetable waxes, fatty alcohols and fatty acids solid at room temperature, glycerides solid at room temperature.

[0066] Among the emulsifiers that can be used in the compositions according to the invention, mention may be made of:

[0067] - fatty esters of alkylpolyglycosides, possibly alkoxylated;

[0068] - alkoxylated fatty esters;

[0069] - fatty chain polyalkylene glycol carbamates;

[0070] - fatty acids, ethoxylated fatty acids, sorbitol fatty acid esters, ethoxylated fatty acid esters, polysorbates, polyglycerol esters, ethoxylated fatty alcohols, sucrose esters, alkylpolyglycosides, sulfated and phosphated fatty alcohols or mixtures of alkylpolyglycosides and fatty alcohols;

[0071] - combinations of emulsifying surfactants chosen from alkylpolyglycosides;

[0072] - combinations of alkylpolyglycosides and fatty alcohols, esters of polyglycerols or polyglycols or polyols.

[0073] Among the surfactants that can be used in the compositions according to the invention, mention may be made of: topically acceptable anionic, cationic, amphoteric or non-ionic surfactants usually used in this field of activity.

[0074] Among the anionic surfactants that can be used in the compositions according to the invention, mention will be made in particular of alkali metal salts, alkaline earth metal salts, ammonium salts, amino acid salts, amino alcohol salts of the following compounds: alkyl ether sulfates, alkyl sulfates, alkylamido ether sulfates, alkylaryl polyether sulfates, monoglyceride sulfates, alpha-olefin sulfonates, paraffin sulfonates, alkyl phosphates, alkyl ether phosphates, alkyl sulfonates, alkylamide sulfonates, alkylaryl sulfonates, alkylcarboxylates, alkyl sulfosuccinates, alkyl ether sulfosuccinates, alkylamide sulfosuccinates, alkyl sulfoacetates, alkyl sarcosinates, acylisethionates, N-acyltaurates, acyllactylates.

[0075] Among the amphoteric surfactants that can be used in the compositions according to the invention, mention may be made of alkylbetaines, alkylamidobetaines, sultaines, alkylamidoalkylsulfobetaines, imidazoline derivatives, phosphobetaines, amphopolyacetates and amphopropionates.

[0076] According to another preferred embodiment, the composition according to the invention is intended for oral administration which can take the form of a nutritional supplement or a dietary preparation.

[0077] For example, said nutritional supplement or dietary preparation may be in the form of granules, tablets, soft capsules, gel caps, suspensions, syrups, ampoules, mouth sprays, drops, walking gums, dissolving powders or solutions.

[0078] In the case of a powder to be dissolved in a liquid, which liquid can then be, for example, water, milk or a milk-based preparation, or even fruit juice.

[0079] Other pharmaceutically and / or food-acceptable agents may be added, such as bulking agents, fluidizers, natural extracts, minerals, trace elements, fatty acids, anti-caking agents, natural oils, flavorings, colorings, acidifiers, thickeners, preservatives and sweeteners.

[0080] Examples of bulking agents include microcrystalline cellulose, potato maltodextrin and magnesium lactate.

[0081] Examples of fluidizers include magnesium silicate, magnesium stearate and colloidal silica.

[0082] Examples of minerals or trace elements include magnesium, iodine, iron, copper, zinc, selenium, chromium, molybdenum, manganese, silicon, vanadium, nickel and tin.

[0083] Examples of amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.

[0084] Examples of fatty acids include unsaturated fatty acids such as omega-3 or omega-6.

[0085] Examples of anti-caking agents commonly used in the food industry include magnesium stearate (E470b), silicon dioxide (E551) and colloidal silica.

[0086] Examples of thickeners include potato starch, hydroxypropylmethylcellulose, citrus pectin, guar gum, carob gum, xanthan gum, agar-agar, konjac, hydrogenated oils and beeswax.

[0087] An example of acidifiers is citric acid.

[0088] Examples of sweeteners include, among others, xylitol, aspartame, glucose syrup, fructooligosaccharide syrup, maltitol powder or syrup, acesulfame potassium, fructooligosaccharide, sodium cyclamate, sucralose.

[0089] Examples of colorants include curcumins (E100), carminic acid (E120), erythrosine (E127), chlorophylls and chlorophyllins (E140), copper-chlorophyll and chlorophyllin complexes (E141), caramel (E1 50), carotenoids (E160), anthocyanins (E163), calcium carbonate (E170), iron oxide and hydroxide (E172) and orcein (E182).

[0090] Examples of preservatives include potassium sorbate, sodium benzoate or ascorbyl palmitate (antioxidant).

[0091] In order to further potentiate the activity obtained by the composition according to the invention, it may be combined with other active ingredients, in particular those known for their anti-aging, firming, restructuring, stimulating, energizing, anti-wrinkle, relaxing, moisturizing, antimicrobial, sebum-regulating, purifying, soothing, relaxing, decontracting, anti-stress, lightening, immunomodulating, cell renewal stimulating, lifting, plumping, complexion radiance improving action, etc.

[0092] According to a second object, the composition described above is used in a method which aims to prevent and / or treat vitiligo in a subject and comprises a step of administering a therapeutically effective amount thereof to a skin surface of said subject.

[0093] By "therapeutically effective amount" is meant an amount sufficient to achieve the desired biological effect.

[0094] The invention will be better understood in light of the following examples, which are given purely for illustrative purposes and are not intended to limit the scope of the invention, defined by the appended claims. Examples

[0095] 1) Ability to induce a drastic decrease in ROS within the mitochondria of keratinocytes of certain specific bacterial extracts

[0096] Primary human keratinocytes (PHK) were isolated from the foreskin of healthy children and cultured in serum-free medium supplemented with bovine pituitary extract (250 pg / mL) and EGF (0.25 ng / mL).

[0097] The NHKs are then seeded onto 6-well plates and incubated in a controlled atmosphere. When the keratinocytes have reached 70 to 80% confluence, the cells are pretreated, or not, for one hour with different components. As a positive control, the NHKs are pretreated for one hour with sinapine (100 pM).

[0098] The plates are then washed 3 times with PBS solution before incubating the pretreated NHKs for one hour in the presence of H2O2 (100 pM) or LPS (100 ng / mL).

[0099] At the end of the incubation, the cells are incubated in a dihydrorhodamine solution (5 pM) for 30 minutes at 37°C, which allows the labeling of ROS, in particular mitochondrial ROS.

[0100] After 3 washes with PBS solution, mitochondrial ROS are quantified by FACS.

[0101] The results are presented in Table 1 below.

[0102] [Table 1]

[0103] Completely unexpectedly, the results showed that extracts of Bifidobacterium longum and Bifidobacterium breve induced a drastic decrease in ROS within the mitochondria of keratinocytes subjected or not to stress, and even to major oxidative stress (H2O2). It should be noted that the observed decrease is specific to the bacterial species used. Indeed, this decrease is not observed with bacterial extracts of the species Bifidobacterium bifidum and Bifidobacterium lactis. The observed decrease in ROS within the mitochondria of keratinocytes with extracts of Bifidobacterium longum and Bifidobacterium breve is much greater than that observed with sinapine.

[0104] It should be noted that additional experiments carried out with plant extracts including sinapine (LIPERFECTION and DETOXOPHANE) showed similar activity of these extracts to that observed with sinapine.

[0105] The results finally show that the combination of extracts of Bifidobacterium longum and Bifidobacterium breve and sinapine makes it possible to obtain a composition exhibiting further increased potential for reducing ROS within the mitochondria of keratinocytes.

[0106] The same tests are carried out on melanocytes.

[0107] 2) Ability to induce a drastic decrease in the expression of proinflammatory cytokines within keratinocytes by certain specific bacterial extracts

[0108] Primary human keratinocytes (PHK) were isolated from the foreskin of healthy children and cultured in serum-free medium supplemented with bovine pituitary extract (250 pg / mL) and EGF (0.25 ng / mL).

[0109] NHKs are then seeded onto 6-well plates and incubated in a controlled atmosphere. When the keratinocytes have reached 70 to 80% confluence, the NHKs are incubated for one hour in the presence of LPS (100 ng / mL) or IFN / TNF (10 ng / ml) for 24 hours.

[0110] At the end of the incubation, the cells are incubated or not in the presence of 100 pg / mL of extracts of different strains of Bifidobacterium for 24 hours. At the end of this incubation period, the supernatants are collected for ELISA assay (TNF alpha, IFN gamma, IL1 beta, CXCL10, CXCL11, and CXCL16). [OR I] The results are presented in Figure 1 which describes the increase in the expression of each of these proinflammatory cytokines (TNF alpha, IFN gamma, IL1 beta, CXCL10, CXCL11, and CXCL16) under these different conditions compared to untreated cells (KHN; reference level 1.0).

[0112] As previously, the results showed that extracts of Bifidobacterium longum and Bifidobacterium breve induced, as for mitochondrial ROS, a drastic decrease in the expression of different pro-inflammatory cytokines within keratinocytes subjected or not to stress. Here again, the observed decrease is specific to the bacterial species used. Indeed, this very significant decrease is not observed with bacterial extracts of the species Bifidobacterium bifidum and Bifidobacterium lactis.

[0113] Again, the same tests are carried out on melanocytes.

[0114] 3) composition according to the invention

[0115] Based on the previous results, a composition was developed comprising an extract of Bifidobacterium longum and a plant extract comprising sinapine so as to prevent the appearance of new lesions or to stabilize them in subjects at an early stage of vitiligo, namely having melanocytes whose mitochondrial function. The details of a composition according to the invention are given in Table 2 below.

[0116] [Table 2]

[0117] Daily application of the composition according to the invention to the skin and, in particular, to areas of skin comprising melanocytes whose mitochondrial function is altered makes it possible to delay the appearance of new lesions and to stabilize existing lesions in subjects at an early stage of vitiligo.

Claims

Demands 1. A composition for use in the prevention and / or treatment, in a subject, of the early stage of vitiligo corresponding to the deregulation of mitochondrial function, of keratinocytes and melanocytes, associated with an abnormally high level of R.OS within these mitochondria, which composition comprises at least one strain of Bifidobacterium longum or Bifidobacterium breve, or a lysate thereof.

2. The composition according to claim 1, characterized in that said composition is intended for the prevention or treatment of a patient suffering from vitiligo.

3. The composition according to any one of the preceding claims, characterized in that said at least one strain of Bifidobacterium longum or Bifidobacterium breve is present in the form of a lysate, preferably said lysate is obtained by ultrasound lysis.

4. The composition according to any one of the preceding claims 5, characterized in that the content of the composition according to the invention in lysate of said at least one strain of Bifidobacterium is between 1 and 20% (by weight relative to the total weight of the composition), preferably it is between 5 and 10%.

5. The composition according to any one of the preceding claims, characterized in that it further comprises at least one antioxidant.

6. The composition according to claim 5, characterized in that said antioxidant is selected from sinapine and its derivatives. REPLACEMENT SHEET (RULE 26) 7. The composition according to the preceding claim, characterized in that the sinapine derivatives have the formula (II): In which - Ri and R3 are chosen independently from a hydrogen atom or an alkyl group at C8, preferably an alkyl group at C4 and, particularly preferably, an alkyl group at C2; and - R2 is chosen independently from a hydrogen atom or an alkyl group in Cl at C8, preferably an alkyl group in Cl at C4 and, particularly preferably, an alkyl group in Cl at C2.

8. The composition according to claim 6, characterized in that the sinapine is in the form of an extract of seeds or germs of a plant belonging to the Brassicaceae family.

9. The composition according to any one of the preceding claims, characterized in that it is intended for topical administration.

10. The composition according to any one of claims 1 to 8, characterized in that it is intended for oral administration. REPLACEMENT SHEET (RULE 26)

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